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Fig. 1. (A) CHL1, CTF8 and CTF4 are required for sister-chromatid cohesion during mitosis. Cycling cultures of wild-type (K10003), chl1{Delta} (K11652), ctf4{Delta} (K11692), ctf8{Delta} (K10576) and trf4{Delta} (K12661) strains expressing Pds1-Myc18 (securin) and carrying chromosome V marked by GFP at the URA3 locus 35 kbp away from the centromere were fixed and stained for DNA and with antibodies against tubulin and the Myc epitope. Fluorescence images of a wild-type (left) and chl1{Delta} (right) metaphase cell showing URA3-GFP (green), tubulin (red) and DNA (blue) in the large frame and securin (white) of the respective cell above in the small inserts. The proportions of securin-positive cells with a bipolar spindle containing one or two URA3-GFP signals in the different strains are given below the images. 100 metaphase cells were scored for each strain in this experiment. (B-E) Chl1 is required during S phase to support sister-chromatid cohesion. A haploid yeast strain (K12131) with chromosome V marked by GFP at the URA3 locus and expressing Pds1-Myc18 (securin) in which the endogenous CHL1 ORF was put under regulation of the Gal1-10 promoter and three HA tags were fused to the N-terminus was arrested with {alpha} factor and released in medium containing raffinose (B) with no addition of galactose, (C) with the addition of galactose at 0 minutes (min) or (D) with addition of galactose at 45 minutes. At the indicated time points, DNA replication was monitored by flow cytometry of DNA content and expression of HA3-Chl1 by immunoblotting with an anti-HA antibody and a loading control (Swi6). (E) Samples from all three cultures taken at 90 minutes were fixed and stained with antibodies against tubulin and the Myc tag of securin to asses the ratio of securin-positive cells with a bipolar spindle containing one or two URA3-GFP signals. 100 metaphase cells were scored for all three cultures in this experiment. (F) Chl1 localizes to the nucleus. Cycling cells of a strain expressing Chl1-Myc18 (K11770) (left) and of an untagged control strain (K8378) (right) were fixed and stained for DNA and with antibodies against tubulin and the Myc epitope. Fluorescence microscopy images of a G1 (left) and metaphase (right) cell showing tubulin (red) and DNA (blue) in the upper panel and anti-Myc staining of the respective cells above in the lower panel.





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